Tuberculosis remains the leading cause of mortality among persons with HIV/AIDS. New approaches to therapy are needed to shorten the duration of therapy, combat drug resistance and kill latent bacteria. Many important advances in understanding the molecular pathogenesis of tuberculosis have been made in the past decade. A major factor in this progress has been the development of molecular genetic tools that allow investigators to manipulate Mycobacterium tuberculosis. While a system for regulated gene expression in mycobacteria was developed several years ago, its relatively high level of basal expression limits its use in many experiments. Defining and characterizing essential genes is a key aspect of defining novel drug targets in M. tuberculosis. The goal of this RO3 small grant application is to adapt an inducible expression system, the positively regulated nitA promoter of Rhodococcus rhodochrous that has minimal basal expression in the absence of induction, for use in mycobacteria. This goal is addressed through Aim 1, in which a) the components of this system will be transferred to a series of replicating and integrating mycobacterial vectors utilizing several selectable markers, b) un-induced expression from this promoter in mycobacteria will be analyzed and the system modified, if needed, to minimize basal expression, and c) the dose response of expression to inducer concentration will be determined. This system will then be used in sub-aim d) to determine whether a gene of interest, such as a potential drug target, is essential. This system will also be tested in sub-aim e) to determine whether it is useful for the expression of full length soluble recombinant mycobacterial proteins in a mycobacterial host, e.g. in M. smegmatis. [unreadable] [unreadable] [unreadable]